Journal of Animal and Feed Sciences, 21, 2012, 217–233
Obtaining farm animal embryos in vitro*
, Ł. Rąpała1
, P. Trzeciak1
, S. Dąbrowski1
and A. Piliszek2
Warsaw University of Life Sciences, Division of Histology and Embryology,
Department of Morphological Sciences, Faculty of Veterinary Medicine
Nowoursynowska 159, 02-776 Warsaw
Institute of Genetics and Animal Breeding, Polish Academy of Sciences,
Experimental Embryology Department
Jastrzębiec, Postępu 1, 05-552 Magdalenka, Poland
(Received 22 March 2012; revised version 21 May 2012; accepted 18 June 2012)
This paper reviews the basic knowledge about obtaining farm animal embryos in vitro
with special focus on differences among species and application of this procedure in the future.
In vitro production of farm animal embryos consists of in vitro maturation (IVM) of oocytes, in
vitro fertilization (IVF) of matured oocytes, and in vitro culture (IVC) of embryos. Oocytes can
be collected from live animals (by laparotomy, laparoscopy, Ovum Pick Up) or from slaughtered
ones (by puncture, sectioning). Usually immature oocytes are isolated, and during IVM they reach
maturity. Matured oocytes are cultured with sperm (IVF), leading to the formation of zygotes. In
the case of fertilization problems (horse, pig), intracytoplasmic sperm injection is used. The zygotes
are usually cultured (IVC) to the morula and blastocyst stages. These embryos can be transferred to
recipients or frozen/vitrified. Offspring have been obtained after transfer of cattle, sheep, goat, pig
and horse embryos. This procedure can be used in animal breeding, biotechnology, medicine, and
KEY WORDS: oocytes, sperm, embryos, in vitro, farm animals
Obtaining farm animal embryos in vitro is a biotechnique used both
commercially (in vitro production, IVP), in basic research, as well as in infertility
Support by COST DPN/DWM/MZ/5670/08/09
Corresponding author: e-mail: firstname.lastname@example.org
218 FARM ANIMALS EMBRYOS OBTAINING IN VITRO
treatment (Gordon, 2004; Thibier, 2005; Betteridge, 2006; Galli and Lazzari, 2008;
Duszewska et al., 2010; Figueirêdo Freitas et al., 2010; Dang-Nguyen et al., 2011;
Zhao et al., 2011). Commercially, it is used in animal breeding and biotechnology,
hence it is called in vitro production of embryos (IETS-www.iets.org). The goal
of this procedure is entirely different in basic research and infertility treatment,
where it plays the same role as the assisted reproduction techniques (ART) used
in humans (Gardner et al., 2011). For this reason it is hard to call this biotechnique
in vitro production of embryos, and instead it is called obtaining embryos in vitro
(Duszewska et al., 2010).
Obtaining farm animal embryos in vitro is a multi-step procedure comprising:
in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro culture (IVC)
(Scheme 1). Offspring of some farm animal species have been obtained after
transfer of embryos obtained in vitro to recipients, including sheep (Gandolfi et
al., 1987), cattle (Lu et al., 1988), pigs (Mattioli et al., 1989), and goats (Crozet et
al., 1993). For many years, obtaining horse embryos in vitro presented the biggest
challenge. In 1991 two foals were born, albeit from naturally matured oocytes that
were fertilized and cultured in vitro (Palmer et al., 1991).
The most important factors responsible for success in obtaining embryos in
vitro are: the source of oocytes and sperm (including the age and physiological
state of female and male), technical factors, and highly skilled laboratory and
veterinary staff. The key technical factors are: media for maturation, fertilization,
and embryo culture (containing water, amino acids, vitamins, hormones,
growth factors, and additives obtained from albumin, serum, etc.), conditions of
maturation, fertilization, and culture (temperature, CO2
level, humidity), and the
timing of oocyte maturation, fertilization, and embryo development (Duran, 2000;
Camargo et al., 2006).
All of these requirements are characteristic of the respective farm animal
species and have been described in detail in the literature: cattle (Hoshi, 2003;
Lonergan, 2007; Lonergan and Fair, 2008), pigs (Abeydeera, 2002; Nagai et al.,
2006; Lonergan and Fair, 2008; Gil et al., 2010; Dang-Nguyen et al., 2011), sheep
and goats (Cognie et al., 2003; Paramio, 2010), and horses (Squires at el., 2003;
Betteridge, 2006, 2007; Hinrichs, 2010).
PROCEDURE OF OBTAINING EMBRYOS IN VITRO FROM FARM ANIMALS
In vitro maturation of oocytes (IVM)
Oocytes are collected from the follicles located in the cortex of the ovary.
There are differences in the localization of the cortex among farm animal species.
In cattle, sheep, goats, and pigs the cortex is the outermost layer, however, in
219DUSZEWSKA A.M. ET AL.
220 FARM ANIMALS EMBRYOS OBTAINING IN VITRO
mares the cortical layer is located inside the ovary, which poses some problems in
Oocytes can be collected from live or slaughtered animals. The methods used
for collection from live animals are: laparotomy (in all species), laparoscopy
(sheep, goats and swine, as well as horses and cattle) and ovum pick up (OPU)
(cattle, horses). OPU entails puncture of ovarian follicles. In sheep and goats,
laparoscopy combined with OPU is a popular method (Pieterse et al., 1991; Carter
et al., 2000; Galli et al., 2001; Abeydeera, 2002; Baldassarre et al., 2002; Cogine
et al., 2003, 2004; Squires et al., 2003; Colleoni et al., 2007; Paramio, 2010).
Oocytes can be collected from females during natural hormonal cycles or after
hormonal stimulation. Pregnant or young females can be used as oocyte donors
(Romaguera, 2011). Mature oocytes (Metaphase II) can be isolated, although in the
majority of cases, immature ones are harvested (Miyano, 2003). The frequency of
oocyte isolation is often a function of ovarian follicle development and maturation,
which is species specific (Hendriksen, 2000; Hirao, 2011; Sirard, 2011).
The most frequently used methods of obtaining material from slaughterhouse
samples are: puncture of an ovarian follicle with a needle connected to a suction
pump (in all species), sectioning of ovaries (mostly in mares), and isolation of
ovarian follicles and subsequent collection of oocytes (in all species) (Abeydeera,
2002; Cogine et al., 2003; Squires et al., 2003).
Immature oocytes are surrounded by several tightly packed layers of granulosa
cells called the corona radiata, with which they form the cumulus-oocyte complex
(COC), and a cluster of cells forming the cumulus oophorus (Abeydeera, 2002;
Cognie et al., 2003; Hoshi, 2003; Nagai et al., 2006; Lonergan, 2007; Lonergan
and Fair, 2008; Gil et al., 2010; Paramio, 2010; Dang-Nguyen et al., 2011). In
mares, oocytes can be surrounded by both tightly packed and dispersed layers of
the corona radiata, but both types of oocytes are competed for maturation (Squires
et al., 2003; Betteridge, 2006; Hinrichs, 2010, 2010a).
Immature oocytes are at the prophase stage of the first meiotic division, namely
in GV (germinal vesicle) stage. Oocyte maturation encompasses simultaneous
nuclear and cytoplasmic changes. The key factors regulating meiosis are MPF
(maturation promoting factor) and MAP(mitogen activated protein kinase), whose
activity is necessary for the oocytes to reach the metaphase II stage (Gilchrist and
In the majority of mammals, resumption of meiosis in vitro is spontaneous
and is commenced by germinal vesicle break down (GVBD) and completed by
reaching the metaphase II stage of meiotic division by the oocyte, the stage when
it can be properly fertilized (Miyano and Manabe, 2007). Cytoplasmic maturation
includes a number of modifications - both morphological (organelle migration,
increase in the number of certain organelles, such as mitochondria, and formation
221DUSZEWSKA A.M. ET AL.
of the cytoskeleton and spindle apparatus) and biochemical (cell cycle regulation,
metabolic processes, accumulation of informational and storage material) (Watson,
The granulosa cells of the corona radiata play an important role in the
maturation of the oocyte, being responsible for contact of the oocyte with the
environment and, therefore, mediating the hormonal and metabolic regulation of
this process (Abeydeera, 2002; Cognie et al., 2003; Hoshi, 2003; Nagai et al.,
2006; Lonergan, 2007; Lonergan and Fair, 2008; Gil et al., 2010; Paramio, 2010;
Dang-Nguyen et al., 2011).
The matured oocyte is characterized by an evenly dispersed corona radiata and
cytoplasm without any signs of degeneration, fragmentation, or vacuolization. In
some farm animal species, the first polar body can be observed, however, in the
majority, it is usually obscured by the opaque oocyte cytoplasm (due to its high
lipid content), therefore, the stage of maturation is determined indirectly on the
basis of dispersion of the granulosa cells of the corona radiata (Abeydeera, 2002;
Cognie et al., 2003; Hoshi, 2003; Nagai et al., 2006; Lonergan, 2007; Lonergan
and Fair, 2008; Gil et al., 2010; Paramio, 2010; Dang-Nguyen et al., 2011).
There are also differences in the timing of oocyte maturation in vitro among
species (Banwell and Thompson, 2008). In cows, sheep, and goats, oocytes reach
maturity (metaphase II), after 24 h culture, and in horse, between 24 and 30 h.
Maturation takes the longest in pig oocytes, where M II is reached within 40-48 h
of culture (Abeydeera, 2002; Cognie et al., 2003; Hoshi, 2003; Nagai et al., 2006;
Lonergan, 2007; Lonergan and Fair, 2008; Gil et al., 2010; Paramio, 2010; Dang-
Nguyen et al., 2011).
In vitro fertilization of matured oocytes
The next step in obtaining embryos in vitro is the fertilization of matured
oocytes. Usually, thawed sperm is used for fertilization, however, since in sheep
and horses problems with freezing sperm occur, fresh sperm is used in these
species (Morrell and Rodriguez-Martinez, 2010) Also, XY sperm separation can
be performed and the obtained sperm used for fertilization (Seidel, 2003; Cran,
Spermatozoa used in in vitro fertilization have to undergo capacitation, which
starts with removal of the glycoprotein coat. This subsequently leads to structural
modifications and changes in the intramembrane protein topography of the head
and tail of the spermatozoon. The changes in the sperm head enable the acrosomal
reaction to occur, whereas changes in the tail lead to hyperactivation (augmented
movement of the sperm). In vitro capacitation is a reversible process, in contrast
to the acrosomal reaction, which is an irreversible process allowing the sperm to
222 FARM ANIMALS EMBRYOS OBTAINING IN VITRO
penetrate the zona pellucida (Tulsiani et al., 2007; Bailey, 2010; Gadella, 2011).
The acrosomal reaction consists of the release of enzymes that allow the sperm
to penetrate the zona pellucida. The entry of sperm leads to oocyte activation,
completion of the II meiotic division resulting in the extrusion of the II polar body.
Subsequently, two pronuclei are formed, DNA replication taking place in each
of them, and the first mitotic division is initiated when the chromosomes of the
oocyte and sperm form a common metaphase plate. Differences among species
can pertain to asynchrony in pronuclei formation and the moment of replication
initiation (Gadella, 2010; Ikawa et al., 2010; Visconti and Florman, 2010; Tulsiani
et al., 2011).
Fertilization in pigs and horses poses many problems, because polyspermy (the
entry of more than one sperm into the oocyte) can often be observed both in vivo
and in vitro. Interestingly, in the case of in vitro matured oocytes, the frequency
of polyspermy is much lower than in in vivo matured oocytes. Polyspermy leads
to creation of polyploid embryos. In some cases triploid embryos can develop
up to the blastocyst stage, however, at later stages they degenerate and undergo
resorption. Because of the high lipid content in the cytoplasm it is not possible to
evaluate the quality of these embryos in the way it is done with human embryos
(Abeydeera, 2002; Cognie et al., 2003; Hoshi, 2003; Nagai et al., 2006; Lonergan,
2007; Lonergan and Fair, 2008; Gil et al., 2010; Paramio, 2010; Dang-Nguyen et
In pigs, polyspermy has been successfully overcome by modification of the
sperm injection (ICSI) (Squires, 1996). In pigs, chromatin decondensation defects
are sometimes observed in the formation of the male pronucleus (Gil et al.,
In horses, in vitro fertilization problems are related to sperm entry into the
oocyte, and, specifically, to the penetration of the zona pellucida (Alm et al., 2001;
Roasa et al., 2007). To date there are only two foals that were obtained with the
use of conventional fertilization (Palmer, 1991). To circumvent this problem, the
ICSI technique, that is, the introduction of the spermatozoon into the cytoplasm
of in vitro matured oocyte, is routinely used (Palermo et al., 1992; Squires et al.,
2003), and has been further modified by the use of piezoICSI (Choi et al., 2002;
Galli et al., 2002).
The ICSI technique can be used in all farm animal species (Garcia-Rosello et
al., 2009), although in cattle, sheep, goats, and pig in vitro fertilization poses no
problems (Goto and Yanagita, 1995; Catt et al., 1996; Kolbe and Holtz, 2000).
In all farm animal species, the timing of fertilization is similar and takes place
18-20 h from insemination. Slight differences are the result of introduction of the
sperm to the oocyte in the ICSI procedure, which can lead to earlier formation of
223DUSZEWSKA A.M. ET AL.
the zygote (Goto and Yanagita, 1995; Catt et al., 1996; Kolbe and Holtz, 2000;
Garcia-Rosello et al., 2009; Nakai et al., 2011).
In vitro culture of embryos
The last stage of obtaining embryos in vitro is their culture, that is the
development of the embryos from zygote to the morula and blastocyst stages. This
encompasses nuclear and cytoplasmic modifications and their mutual interactions,
changes in embryo metabolism, embryonic genome activation, modifications
of gene expression, formation of the morula and compaction of blastomeres,
formation of the blastocyst with a characteristic structure named the blastocyst
cavity that arises as a result of cavitation, and formation of additional structures
such as the capsule in the horse embryos (Abeydeera, 2002; Cognie et al., 2003;
Hoshi, 2003; Nagai et al., 2006; Lonergan, 2007; Lonergan and Fair, 2008; Gil et
al., 2010; Paramio, 2010; Dang-Nguyen et al., 2011). The capsule is a structure
located between trophoblast and zona pellucida, which is generated by the embryo.
Its role is to protect the embryo and it is found in embryos obtained both in vitro
and in vivo (Tremoleda et al., 2003).
One of the most important processes that take place during embryonic
development both in vitro and in vivo is the acquisition of control by the
embryonic genome - the maternal-zygotic transition (MET) (Vigneault et al.,
2004). Activation of the zygotic genome takes place at the 4-blastomere stage in
pigs, and the 8-16 blastomere stage in cattle, sheep, goats, and horses. Up until the
moment of embryonic genome activation, embryonic development is based on the
material stored during oocyte maturation (Sirard, 2010).
Differences occur among species in in vitro obtained blastocyst morphology
that concern the size of the embryos and the number of blastomeres forming the
inner cell mass (ICM) and the trophectoderm. The significant differences in the
number of blastomeres in horse embryos between blastocysts obtained in vivo
(1761 blastomeres) and in vitro (255 blastomeres) deserve special attention. In
other species the observed differences are not that great (Pomar et al., 2005).
The duration of embryonic development up to the blastocyst stage is
characteristic of a given species and is connected with the length and number
of consecutive cell cycles. Pig embryos have the shortest development time,
reaching blastocyst stage in only 144 h, while cattle, ovine, and goat blastocysts
are formed within 168 h. Equine embryos develop the longest (168-192 h of
culture) (Abeydeera, 2002; Cognie et al., 2003; Hoshi, 2003; Nagai et al., 2006;
Lonergan, 2007; Lonergan and Fair, 2008; Gil et al., 2010; Paramio, 2010; Dang-
Nguyen et al., 2011).
224 FARM ANIMALS EMBRYOS OBTAINING IN VITRO
After the transfer of farm species embryos obtained in vitro to recipients,
the percentage of offspring is lower than in case of embryos obtained in vivo
(Abeydeera, 2002; Cognie et al., 2003; Hoshi, 2003; Nagai et al., 2006; Lonergan,
2007; Lonergan and Fair, 2008; Gil et al., 2010; Paramio, 2010; Dang-Nguyen et
The percentage of calving, kidding, or foaling after transfer of in vivo and in
vitro embryos is, respectively, cattle, 70 vs 45; sheep, 75 vs 30; horses, 75 vs 20;
goats, 75 vs 30; pigs, 60 vs 5. In the case of pigs the percentage is extremely low
(Pomar et al., 2005).
Embryos obtained in vitro can be frozen or vitrified and later transferred to
recipients. Their developmental abilities after transfer are much lower, however,
than non-frozen embryos obtained in vitro. In the majority of farm animal species,
progeny is obtained after transfer of embryos frozen at the early or mature
blastocyst stage. On the other hand, much less offspring can be obtained after
transfer of frozen, morula-stage in vitro embryos. Great hopes lie in vitrification,
which allows for higher survival rates of embryos and, moreover, enables obtaining
more progeny after their transfer (Dobrinsky, 2002; Vajta and Kuwayama, 2006).
CRYOPRESERVATION OF EMBRYOS AND OOCYTES
Cryopreservation of oocytes and embryos is performed in two ways: slow
freezing and vitrification. Slow freezing is a conventional method based on a low
concentration of cryoprotectant and slow temperature reduction. Vitrification is
an alternative method characterized by a high cryoprotectant concentration and
ultra-rapid cooling rates, which makes it possible to avoid water precipitation,
thus preventing intracellular ice crystal formation. In this case, the short time
of vitrification and elimination of costly programmable freezing equipment is
of great importance. There are many modifications of freezing and vitrification
procedures (Vajta and Kuwayama, 2006; Ledda et al., 2007; Pereira and Marques,
2008; Prentiz and Anzar, 2011; Saragusty and Arav, 2011).
Interspecific differences exist in cryopreservation resistance of both oocytes
and embryos. Oocytes can be cryopreserved at the GV stage and in metaphase
II, but compared with embryos, oocytes are much more sensitive. As a result of
cryoconservation they develop morphological and functional defects (Martins
et al., 2005; Contreras et al., 2008; Pereira and Marques, 2008), therefore, the
to the granulosa cells surrounding them; because of their role in oocyte maturation
they can not be removed. Further problems with oocyte cryopreservation are
225DUSZEWSKA A.M. ET AL.
related to the morphology of the metaphase II oocyte cell membrane and cortical
granules and spindle apparatus (Pereira and Marques, 2008).
With the use of the conventional, slow method of freezing oocytes, offspring
have been obtained from many species, such as cattle (Otoi et al., 1996), sheep
(Woods et al., 2004), horse (Maclellan et al., 2002). After vitrification, offspring
have been obtained in cattle (Sripunya et al., 2010), pigs (Liu, et al., 2008), sheep
(Succu et al., 2007), and horse (Bogliolo et al., 2006).
Cryopreservation of farm animal embryos obtained in vitro does not pose as
many problems as that of oocytes, however, even in that case the efficiency of
freezing depends on the species, developmental stage of the embryos, and their
origin (in vitro or in vivo), as well as the method of cryoconservation and choice
of cryoprotectant. Cattle and sheep embryos are the most resistant, the least - pig
and horse embryos (Pereira and Marques, 2008). In the 1980s and 1990s, progeny
was obtained after freezing and thawing of farm animal embryos. Also after
vitrification of in vitro embryos, progeny of many farm animals has been obtained
(Dobrinsky, 2002; Vajta and Kuwayama, 2006; Saragusty and Arav, 2011).
DEVELOPMENT POTENTIAL OF EMBRYOS OBTAINED IN VITRO
The aim of the procedure of in vitro embryo production is to obtain a large
number and high quality of blastocysts. The number of embryos obtained depends
on the number of collected oocytes and their quality. On the other hand, the
quality of the blastocysts also depends on the conditions during oocyte maturation
and embryo culture and on the bull’s influence. Both quality and quantity of
blastocysts determines the number of offspring and its healthiness after transfer of
those embryos into recipients (van Wagtendonk-de Leeuw et al., 2000; Rizos et
al., 2002; Lonergan et al., 2003; McEvoy et al., 2006; Hansen et al., 2010; Lazzari
et al., 2010; Romaguera et al., 2010).
The development potential of embryos obtained in vitro is lower in comparison
with embryos obtained in vivo due to suboptimal conditions during oocyte
maturation, their subsequent fertilization, and embryonic development (Wright
and Ellington, 1995; Young et al., 1998; Lonergan et al., 2006; Metwally and
Evaluation of embryos is based mainly on the timing of mitotic cleavage
and on embryo morphology. In the morphological aspect, during the oocyte and
early embryonic development stages, one can observe inhibition of development,
fragmentation, vacuolization, and lysis. Analysis of embryonic development
indicates that in all species of farm animals, high mortality occurs at the early
stages of in vitro development. Among the types of death of single blastomeres,
and sometimes of all the blastomeres of the embryo, the most prominent are
226 FARM ANIMALS EMBRYOS OBTAINING IN VITRO
necrosis and senescence. Far less frequent is programmed cell death (apoptosis),
which can be observed mainly in horse embryos. More recently other types of cell
death have been attracting greater attention, specifically, autophagy and mitotic
catastrophe (Okada et al., 2004). One can not exclude other, so far unknown, types
of cell death (Betts et al., 2001; Okada et al., 2004).
These events do not always lead to embryo death, sometimes they will only
cause perturbations of development, which can become manifest much later, after
and occurrence of disorders are: changes of metabolism during oocyte maturation
and embryo development, anomalous activation of the embryonic genome and
defective gene expression, chromosomal aberrations (polyploidy, aneuploidy,
mixploidy) (van Wagtendonk-de Leeuw et al., 2000). Epigenetic modifications, i.e.
can occur in vitro present an interesting problem. Epigenetic modifications during
maturation and embryonic development, and probably also during fertilization,
can alter preimplantation, postimplantation, and also postnatal development of
individuals. This can lead to various disorders, including: hydrocephalus, brain
hypoplasia or damage, abnormal limb development, heart and liver enlargement,
respiratory problems, as well as large offspring syndrome (LOS). Epigenetic
modifications can lead to the death of individuals in the pre- and postnatal period
(resorption, abortion, or death shortly after birth) (Young et al., 2000; Weaver et
al., 2009; Bartolomei and Ferguson-Smith, 2011; Li and Sasaki, 2011).
It should be stressed, however, that in most cases the progeny obtained after
transfer of the farm animal embryos obtained in vitro is indistinguishable both
anatomically and physiologically from that obtained under natural conditions.
APPLICATION OF PROCEDURES FOR OBTAINING EMBRYOS IN VITRO
FROM FARM ANIMALS
Obtaining livestock embryos in vitro has applications in animal breeding,
biotechnology, medicine, and basic research (Betteridge, 2006; Hansen, 2006;
Galli and Lazzari, 2008; Duszewska et al., 2010).
In animal breeding the procedure of obtaining embryos in vitro can be used
as an alternative technique to insemination or a multiple ovulation and embryo
transfer (MOET) programme, which consists of inducing multiple ovulations and
transferring embryos to recipients. It is possible to use females only as oocyte
donors without the burden of the further stages of this procedure. Immature or
pregnant females can also be used as oocyte donors. Oocytes and embryos can be
frozen or vitrified, which enables banking them (Gardner et al., 2011).
227DUSZEWSKA A.M. ET AL.
Obtaining embryos in vitro is of the greatest importance in biotechnology
because this technique facilitates embryonic and somatic cloning, creating
transgenic individuals and their clones, and also creating chimeras. The great
hopes of human medicine lie in use of pigs as organ donors in xenotransplantation
(Duszewska et al., 2010).
Basic research in both oocyte maturation, oocyte fertilization and early
embryonic development is an important field that makes use of embryos obtained
in vitro. Early development of bovine embryos is similar to human embryos and,
therefore, cattle is considered to be a model species (Niemann et al., 2000).
In recent years more attention has been paid to the application of this
biotechnique in infertility treatment in farm animals, where it plays the same
role as assisted reproduction techniques (ART) used in humans. There are many
indications connected with farm animal infertility for the application of the in
vitro embryo obtaining procedure, including ICSI. Moreover, in special cases, the
microsurgical epididymal sperm aspiration (MESA) or testicular sperm aspiration
(TESA) (testicle biopsy) procedures can be applied, with subsequent use of ICSI
(Greve and Callesen, 2005; Duszewska et al., 2010).
Recommendations for use of procedures for obtaining embryos in vitro can
1. Oviductal obstruction; in this case females are used only as oocyte donors.
2. The use of females only at the most desired moment, which is facilitated by
cryoconservation of gametes and embryos, that is, freezing and vitrification
of immature and mature oocytes and embryos, which enables banking them
similarly to freezing sperm, and for subsequent transfer of embryos to the
mother or surrogate mother.
3. Sex-linked genetic aberrations; in this case it is possible to regulate the sex in
a herd by use of sexed semen for in vitro fertilization.
4. Carrier-state of lethal genes can be detected at the early stages of embryonic
development and makes it possible to eliminate those embryos from the
5. Polyspermy, that is, the penetration of more than one sparmatozoa into
the oocyte, generates polyploidy, e.g., triploid embryos, which develop
only until the blastocyst stage and undergo resorption at the later stages of
6. Immunological infertility.
7. Insufficient number of motile spermatozoa (oligospermia) or excessive
thickness of the zona pellucida, which prevents sperm penetration, are
indications for ICSI.
228 FARM ANIMALS EMBRYOS OBTAINING IN VITRO
8. Preimplantation genetic diagnosis (PGD) can supplement prenatal diagnostics,
which is a separate field in human medicine in infertility treatment. Among
the many directions of PGD, the most important include diagnostics of
chromosomal aberrations, including hereditary diseases.
Thanks to progress in many fields of science it has become possible to
treat infertility not only in humans, but also in farm animals by use of assisted
reproduction techniques and techniques used in biotechnology.
The procedure of obtaining farm animal embryos in vitro is commonly used
and is considered to be one of the most important biotechniques in reproduction
since it combines animal breeding with biotechnology as well as human and
veterinary medicine. Obtaining embryos in vitro is of the greatest importance in
biotechnology because this technique facilitates embryonic and somatic cloning,
creating transgenic individuals, their cloning, and also creating chimeras. The great
hopes of human medicine lie in use of pigs as organ donors in xenotransplantation.
Obtaining farm animal embryos in vitro may be used in infertility treatment.
Abeydeera L.R., 2002. In vitro production of embryos in swine. Theriogenology 57, 257-273
Alm H., Torner H., Blottner S., Nürnberg G., Kanitz W., 2001. Effect of sperm cryopreservation
and treatment with calcium ionophore or heparin on in vitro fertilization of horse oocytes.
Theriogenology 56, 817-829
Bailey J.L., 2010. Factors regulating sperm capacitation. Syst. Biol. Reprod. Med. 56, 334-348
Baldassarre H., Wang B., Kafidi N., Keefer C., Lazaris A., Karatza‘s C.N., 2002. Advances in the
production and propagation of transgenic goats using laparoscopic ovum pick-up and in vitro
embryo production technologies. Theriogenology 57, 275-284
Banwell K.M., Thompson J.G., 2008. In vitro maturation of mammalian oocytes: outcomes and
consequences. Semin. Reprod. Med. 26, 162-174
Bartolomei M.S., Ferguson-Smith A.C., 2011. Mammalian genomic imprinting. CSH Perspect.
Betteridge K.J., 2006. Farm animal embryo technologies: achievements and perspectives.
Theriogenology 65, 905-913
Betteridge K.J., 2007. Equine embryology: an inventory of unanswered questions. Theriogenology
Betts D.H., King W.A., 2001. Genetic regulation of embryo death and senescence. Theriogenology
Bogliolo L., Ariu F., Rosati I. et al., 2006. Vitrification of immature and in vitro matured horse
oocytes. Reprod. Fert. Develop. 18, 149-150
229DUSZEWSKA A.M. ET AL.
Camargo L.S.A., Viana J.H.M., Sá W.F., Ferreira A.M., Ramos A.A., Vale Filho V.R., 2006. Factors
influencing in vitro embryo production. Anim. Reprod. 3, 19-28
Carter J.A., Meintjes M., Bellow M.S., Cochran R., Graff K., Godke R.A., 2000. Oocyte aspiration
for in vitro embryo production in farm animals. Louisiana Agr. Mag. 43, 8-10
Catt S.L., Catt J.W., Gomez M.C., Maxwell W.M., Evans G., 1996. Birth of a male lamb derived
from an in vitro matured oocyte fertilised by intracytoplasmic injection of a single presumptive
male sperm. Vet. Rec. 139, 494–495
Choi Y.H., Love C.C., Love L.B., Varner D.D., Brinsko S., Hinrichs K., 2002. Developmental
competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic
sperm injection with fresh or frozen-thawed spermatozoa. Reproduction 123, 455-465
Cognié Y., Poulin N., Locatelli Y., Mermillod P., 2004. State-of-the-art production, conservation and
transfer of in-vitro-produced embryos in small ruminants. Reprod. Fert. Develop. 16, 437-45
Cognie Y., Baril G., Poulin N., Mermillod P., 2003. Current status of embryo technologies in sheep
and goat. Theriogenology 59, 171-188
Colleoni S., Barbacini S., Necchi D., Duchi R., Lazzari G., Galli C., 2007. Application of ovum
pick-up, intracytoplasmic sperm injection and embryo culture in equine practice. Proc. Amer.
Assn. Equine Pract. 53, 554-559
Contreras D.A., Galina C.S., Avila J.G., Aspron M.P., Moreno-Mendoza N., 2008. A system to
evaluate the quality of frozen embryos through short-term culture. Anim. Reprod. Sci. 106,
Cran D.G., 2007. XY sperm separation and use in artificial insemination and other ARTs. Soc.
Reprod. Fertil. Suppl. 65, 475-491
Crozet N., Smedt V., Ahmed-Ali M., Sevellec C., 1993. Normal development following in vitro
oocyte maturation and fertilization in the goat. Theriogenology 39, 206 (Abstr.)
Dang-Nguyen T.Q., Somfai T., Haraguchi S., Kikuchi K., Tajima A., Kanai Y., Nagai T., 2011.
In vitro production of porcine embryos: current status, future perspectives and alternative
applications. Anim. Sci. J. 82, 374-382
Dobrinsky J.R., 2002. Advancements in cryopreservation of domestic animal embryos.
Theriogenology 57, 285-302
Duran D., 2000. Technical aspect of in vitro embryo production. Food Fertil. Technol. Center www.
DuszewskaA.M., Trzeciak P., CompaA., Rąpała Ł., 2010. Selected issues concerning biotechnology
of farm animals breeding - a review. Anim. Sci. Pap. Rep. 28, 295-306
Figueirêdo Freitas V.J., Melo L.M., 2010. In vitro embryo production in small ruminants. Rev. Bras.
Zootec. 39, 409-413
Gadella B.M., 2010. Interaction of sperm with the zona pellucida during fertilization. Soc. Reprod.
Fertil. Suppl. 67, 267-287
Gadella B.M., Evans J.P., 2011. Membrane fusions during mammalian fertilization. Adv. Exp. Med.
Biol. 713, 65-80
Galli C., Crotti G., Notari C., Turini P., Duchi R., Lazzari G., 2001. Embryo production by ovum
pick up from live donors. Theriogenology 55, 1341-1357
Galli C., Crotti G., Turini P., Duchi R., Mari G., Zavaglia G., Duchamp G., Daels P., Lazzari G.,
2002. Frozen- thawed embryos produced by Ovum Pick Up of immature oocytes and ICSI are
capable to establish pregnancies in the horse. Theriogenology 58, 705-708
Galli C, Lazzari G., 2008. The manipulation of gametes and embryos in farm animals. Reprod.
Domest. Anim. 43, 1-7
Gandolfi F., Moor R.M., 1987. Stimulation of early embryonic development in the sheep by co-
culture with oviduct epithelial cells. J. Reprod. Fertil. 81, 23-28
230 FARM ANIMALS EMBRYOS OBTAINING IN VITRO
Gardner D.K., Boros R.M.R., Falcone T., 2011. Human Assisted Reproductive Technology: Future
Trends in Laboratory and Clinical Practice. Cambridge Medicine, pp. 336
García-Roselló E., García-Mengual E., Coy P., Alfonso J., Silvestre M.A., 2009. Intracytoplasmic
sperm injection in livestock species: an update. Reprod. Domest. Anim. 44, 143-151
Gil M.A., Cuello C., Parrilla I., Vazquez J.M., Roca J., Martinez E.A., 2010. Advances in swine in
vitro embryo production technologies. Reprod. Domest. Anim. 45, 40-48
Gilchrist R.B., Thompson J.G., 2007. Oocyte maturation: emerging concepts and technologies to
improve developmental potential in vitro. Theriogenology 67, 6-15
Gordon I.R., 2004. Reproductive Technologies in Farm Animals. CABI, pp. 332
Goto K., Yanagita K., 1995. Normality of calves obtained by intracytoplasmic sperm injection.
Hum. Reprod. 10, 1554 (Abstr.)
Greve T., Callesen H., 2005. Embryo technology: implications for fertility in cattle. Rev. Sci. Tech.
Hansen P.J., 2006. Realizing the promise of IVF in cattle--an overview. Theriogenology 65,
Hansen P.J., Block J., Loureiro B., Bonilla L., Hendricks K.E., 2010. Effects of gamete source and
culture conditions on the competence of in vitro-produced embryos for post-transfer survival in
cattle. Reprod. Fert. Develop. 22, 59-66
Hendriksen P.J., Vos P.L., Steenweg W.N., Bevers M.M., Dieleman S.J., 2000. Bovine follicular
development and its effect on the in vitro competence of oocytes. Theriogenology 53, 11-20
Hinrichs K., 2010. In vitro production of equine embryos: state of the art. Reprod. Domest. Anim.
Hinrichs K., 2010a. The equine oocyte: factors affecting meiotic and developmental competence.
Mol. Reprod. Dev. 77, 651-661
Hirao Y., 2011. Conditions affecting growth and developmental competence of mammalian oocytes
in vitro. Anim. Sci. J. 82, 187-197
Hoshi H., 2003. In vitro production of bovine embryos and their application for embryo transfer.
Theriogenology 59, 675-685
Ikawa M., Inoue N., Benham A.M., Okabe M., 2010. Fertilization: a sperm’s journey to and
interaction with the oocyte. J. Clin. Invest. 120, 984-94
Kolbe T., Holtz W., 2000. Birth of a piglet derived from an oocyte fertilized by intracytoplasmic
sperm injection (ICSI). Anim. Reprod. Sci. 64, 97-101
Lazzari G., Colleoni S., Lagutina I., Crotti G., Turini P., Tessaro I., Brunetti D., Duchi R., Galli C.,
2010. Short-term and long-term effects of embryo culture in the surrogate sheep oviduct versus
in vitro culture for different domestic species. Theriogenology 73, 748-757
Ledda S., Bogliolo L., Succu S., Ariu F., Bebbere D., Leoni G.G., Naitana S., 2007. Oocyte
cryopreservation: oocyte assessment and strategies for improving survival. Reprod. Fert.
Develop. 19, 13-23
Li Y., Sasaki H., 2011. Genomic imprinting in mammals: its life cycle, molecular mechanisms and
reprogramming. Cell Res. 21, 466-473
Liu Y., Du Y., Lin L. et al., 2008. Comparison of efficiency of open pulled straw (OPS) and cryotop
vitrification for cryopreservation of in vitro matured pig oocytes. Cryo-Lett. 29, 315-320
Lonergan P., 2007. State-of-the-art embryo technologies in cattle. Soc. Reprod. Fertil. Suppl. 64,
Lonergan P., FairT., 2008. In vitro-produced bovine embryos: dealing with the warts.Theriogenology
Lonergan P., FairT., Corcoran D., EvansA.C., 2006. Effect of culture environment on gene expression
and developmental characteristics in IVF-derived embryos. Theriogenology 65, 137-152
231DUSZEWSKA A.M. ET AL.
Lonergan P., Rizos D., Gutierrez-Adan A., Fair T., Boland M.P., 2003. Oocyte and embryo quality:
effect of origin, culture conditions and gene expression patterns. Reprod. Domest. Anim. 38,
Lu K.H., Gordon I., Chen H.B., Gallagher M., McGovern H., 1988. Birth of twins after transfer of
cattle embryos produced by in vitro techniques. Vet. Rec. 122, 539-540
Maclellan L.J., Carnevale E.M., Coutinho da Silva M.A., Scoggin C.F., Bruemmer J.E., Squires
E.L., 2002. Pregnancies from vitrified equine oocytes collected from superstimulated and non-
stimulated mares. Theriogenology 58, 911-919
Martins R.D., Costa E.P., Chagas J.S.C., Ignácio F.S., Torres C.A.A., McManus C., 2005. Effects of
vitrification of immature bovine oocytes on in vitro maturation. Anim. Reprod. 2, 128-134
Mattioli M., Bacci M.L., Galeati G., Seren E., 1989. Developmental competence of pig oocytes
matured and fertilized in. vitro. Theriogenology 31, 1201-1207
McEvoy T.G., Alink F.M., Moreira V.C., Watt R.G., Powell K.A., 2006. Embryo technologies
and animal health - consequences for the animal following ovum pick-up, in vitro embryo
production and somatic cell nuclear transfer. Theriogenology 65, 926-942
Metwally M., Ledger W.L., 2011. Long-term complications of assisted reproductive technologies.
Hum. Fertil. (Camb). 14, 77-87
Miyano T., 2003. Bringing up small oocytes to eggs in pigs and cows. Theriogenology 59, 61-72
Miyano T., Manabe N., 2007. Oocyte growth and acquisition of meiotic competence. Soc. Reprod.
Fertil. Suppl. 63, 531-538
Morrell J.M., Rodriguez-Martinez H., 2010. Practical applications of sperm selection techniques as
a tool for improving reproductive efficiency. Vet. Med. Int. pii: 894767
Nagai T., Funahashi H., Yoshioka K., Kikuchi K., 2006. Up date of in vitro production of porcine
embryos. Front. Biosci. 1, 2565-2573
Nakai M., Kashiwazaki N., Ito J., Maedomari N., Ozawa M., Shino M., Noguchi J., Kaneko
H., Kikuchi K., 2011. Factors affecting fertilization and embryonic development during
intracytoplasmic sperm injection in pigs. J. Reprod. Dev. 57, 183-187
Niemann H., Wrenzycki C., 2000. Alterations of expression of developmentally important genes
in preimplantation bovine embryos by in vitro culture conditions: implications for subsequent
development. Theriogenology 53, 21-23
Okada H., Mak T.W., 2004. Pathways of apoptotic and non-apoptotic death in tumor cells. Nat. Rev.
Cancer 4, 592-603
Otoi T., Yamamoto K., Koyama N., Tachikawa S., Suzuki T., 1996.Afrozen-thawed in vitro-matured
bovine oocyte derived calf with normal growth and fertility. J. Vet. Med. Sci. 58, 811-813
Palermo G., Joris H., Devroey P., Van Steirteghem A.C., 1992. Pregnancies after intracytoplasmic
injection of single spermatozoon into an oocyte. Lancet 340, 17-18
Palmer E., Bézard J., Magistrini M., Duchamp G., 1991. In vitro fertilization in the horse. A
retrospective study. J. Reprod. Fertil. 44, Suppl., 375-384
Paramio M.T., 2010. In vivo and in vitro embryo production in goats. Small Ruminant Res. 89,
Pereira R.M., Marques C.C., 2008. Animal oocyte and embryo cryopreservation. Cell Tissue Bank
Pieterse M.C., Vos P.L., Kruip T.A., Wurth Y.A., van Beneden T.H., Willemse A.H., Taverne M.A.,
1991. Transvaginal ultrasound guided follicular aspiration of bovine oocytes. Theriogenology
Pomar F.J., Teerds K.J., Kidson A., Colenbrander B., Tharasanit T., Aguilar B., Roelen B.A.J., 2005.
Differences in the incidence of apoptosis between in vivo and in vitro produced blastocysts of
farm animal species: a comparative study. Theriogenology 63, 2254-2268
232 FARM ANIMALS EMBRYOS OBTAINING IN VITRO
Prentice J.R, Anzar M., 2011. Cryopreservation of mammalian oocyte for conservation of animal
genetics. Vet. Med. Int. doi:10.4061/2011/146405
Rizos D., Ward F., Duffy P., Boland M.P., Lonergan P., 2002. Consequences of bovine oocyte
maturation, fertilization or early embryo development in vitro versus in vivo: Implication for
blastocysts yield and blastocysts quality. Mol. Reprod. Dev. 61, 234-248
Roasa L.M., Choi Y.H., Love C.C., Romo S., Varner D.D., Hinrichs K., 2007. Ejaculate and type
of freezing extender affect rates of fertilization of horse oocytes in vitro. Theriogenology 68,
Romaguera R., Casanovas A., Morató R., Izquierdo D., Catalá M., Jimenez-Macedo A.R., Mogas T.,
Paramio M.T., 2010. Effect of follicle diameter on oocyte apoptosis, embryo development and
chromosomal ploidy in prepubertal goats. Theriogenology 74, 364-373
Romaguera R., Moll X., Morató R., Roura M., Palomo M.J., Catalá M.G., Jiménez-Macedo A.R.,
Hammami S., Izquierdo D., Mogas T., Paramio M.T., 2011. Prepubertal goat oocytes from large
follicles result in similar blastocyst production and embryo ploidy than those from adult goats.
Theriogenology 76, 1-11
Saragusty J., Arav A., 2011. Current progress in oocytes and embryo cryopreservation by slow free
and vitrification. Reproduction 141, 1-19
Seidel G.E., 2003. Sexing mammalian sperm - intertwining of commerce, technology, and biology.
Anim. Reprod. Sci. 79, 145-156
Sirard M.A., 2010. Activation of the embryonic genome. Soc. Reprod. Fertil., Suppl. 67, 145-158
Sirard M.A., 2011. Follicle environment and quality of in vitro matured oocytes. J. Assist. Reprod.
Genet. 28, 483-488
Squires E.L., Carnevale E.M., McCue P.M., Bruemmer J.E., 2003. Embryo technologies in the
horse. Theriogenology 59, 151-170
Squires E.L., Wilson, J.M., Kato, H., Blaszczyk, A., 1996. A pregnancy after intracytoplasmic sperm
injection into equine oocytes matured in vitro. Theriogenology 45, 306 (Abstr.)
Sripunya N., Somfai T., Inaba Y., Nagai T., Imai K., Parnpai R., 2010. A comparison of cryotop and
solid surface vitrification methods for the cryopreservation of in vitro matured bovine oocytes.
J. Reprod. Dev. 56, 176-181
Succu S., Leoni G.G., Bebbere D. et al., 2007. Vitrification devices affect structural and molecular
status of in vitro matured ovine oocytes. Mol. Reprod. Dev. 74, 1337-1344
Thibier M., 2005. The zootechnical application of biotechnology in animal reproduction: current
methods and perspectives. Reprod. Nutr. Develop. 45, 235-242
Tremoleda J.L., Stout T.A., Lagutina I., Lazzari G., Bevers M.M., Colenbrander B., Galli C., 2003.
Effects of in vitro production on horse embryo morphology, cytoskeletal characteristics, and
blastocyst capsule formation. Biol. Reprod. 69, 1895-1906
Tulsiani D.R., Abou-Haila A., 2011. Molecular events that regulate mammalian fertilization.
Minerva Ginecol. 63, 103-118
Tulsiani D.R., Zeng H.T.,Abou-HailaA., 2007. Biology of sperm capacitation: evidence for multiple
signalling pathways. Soc. Reprod. Fertil. Suppl. 63, 257-272
Vajta G. and Kuwayama M., 2006. Improving cryopreservation systems. Theriogenology 65, 236-
van Wagtendonk-de Leeuw A.M., Mullaart E., de Roos A.P.W., Merton J.S., den Daas J.H.G., Kemp
B., de Ruigh L., 2000. Effects of different reproduction techniques: AI, MOET or IVP on health
and welfare of bovine offspring. Theriogenology 53, 575-597
Vigneault C., Mcgraw S., Massicotte L., Sirard M.A., 2004. Transcription factor expression patterns
in bovine in vitro-derived embryos prior to maternal-zygotic transition. Biol. Reprod. 70, 1701-
233DUSZEWSKA A.M. ET AL.
Watson A.J., 2007. Oocyte cytoplasmic maturation: a key mediator of oocyte and embryo
developmental competence. J. Anim. Sci. 85, Suppl. E, E1-E3
Weaver J.R., Susiarjo M., Bartolomei M.S., 2009. Imprinting and epigenetic changes in the early
embryo. Mamm. Genome 20, 532-543
Woods E.J., Benson J.D., Agca Y., Critser J.K., 2004. Fundamental cryobiology of reproductive
cells and tissues. Cryobiology 48, 146-156
Wright R.W., Ellington J., 1995. Morphological and physiological differences between in vivo- and
in vitro-produced preimplantation embryos from livestock species. Theriogenology 44, 1167-
Young L.E., Fairburn H.R., 2000. Improving the safety of embryo technologies: possible role of
genomic imprinting. Theriogenology 53, 627-648
Young L.E., Sinclair K.D., Wilmut I., 1998. Large offspring syndrome in cattle and sheep. Rev.
Reprod. 3, 155-163
Zhao Y., Brezina P., Hsu C.C., Garcia J., Brinsden P.R., Wallach E., 2011. In vitro fertilization: four
decades of reflections and promises. Biochim. Biophys. Acta 1810, 843-852